In general, a superoxide anion radical (O2−.) which is a reactive oxygen species is produced in vivo by the oxidation of xanthine as well as hypoxanthine to uric acid by xanthine/xanthine oxidase (XOD) and the reduction of the enzyme by hemoglobin, and the like, and has an important role in association with the in vivo synthesis of a physiologically active substance, a sterilizing effect, an aging phenomenon and the like. On the other hand, it is alleged that various reactive oxygen species derived from the superoxide anion radical cause various diseases such as a cancer. Therefore, the measurement of a concentration of reactive oxygen species including an in vivo superoxide anion radical is considered to be important to specify the various diseases.
The superoxide anion radical, if no substrate exists, is converted into hydrogen peroxide and oxygen molecule (O2) by a disproportionating reaction, as indicated in an equation (1). The disproportionating reaction comprises the production of HO2. by the addition of proton to the superoxide anion radical, the production of hydrogen peroxide and oxygen molecule by the reaction of HO2. and oxygen molecule, and the production of hydrogen peroxide and oxygen molecule by the collision of HO2. and HO2. (equations (1) to (4)).2H++2O2−.→H2O2+O2  (1)H++O2−.→HO2.  (2)HO2.+O2−.+H+→H2O2+O2  (3)HO2.+HO2−.→H2O2+O2  (4)
In this reaction system, the superoxide anion radical acts as an electron acceptor (an oxidizing agent), an electron donor (a reducing agent) and a hydrogen ion acceptor (a base), and an attempt has been made to measure a concentration of a superoxide anion radical, utilizing the natures of first two of the donor and the acceptor. For example, an attempt has been made to measure a concentration of a superoxide anion radical, utilizing the reaction of conversion from ferri-cytochrome c (trivalent) to ferro-cytochrome c (divalent), the reaction of production of formazane blue from nitro-tretrazolium blue (NBT) and the reducing reaction of tetranitromethane (TNM). All of these measurements have been made by an in vitro measuring process.
On the other hand, reviews have been made for a process for quantitatively detecting a concentration of an in vivo superoxide anion radical. For example, McNeil et al, Tatiov et al and Cooper et al have reported that a concentration of a superoxide anion radical can be electrochemically detected by an enzyme electrode (cytochrome c-fixed electrode) fabricated by decorating a surface of an electrode of gold or platinum with N-acetyl cysteine, and S—Au bonding and fixing, to the resulting surface, a protein such as cytochrome c which is a metal protein based on an iron complex called heme for oxidation-reduction (see the following Documents 1 to 3).
Document 1                C. J. McNeil et al. Free Radical Res. Commun., 7, 89 (1989)        
Document 2                M. J. Tariov et al. J. Am. Chem. Soc. 113, 1847 (1991)        
Document 3                J. M. Cooper, K. R. Greenough and C. J. McNeil, J. Electroan al. Chem., 347, 267 (1993)        
The measuring principle of this detecting method is as follows: Cytochrome c (trivalent) (cyt.c (Fe3+)) is reduced into cytochrome c (divalent) (cyt.c (Fe2+)) by reacting with a superoxide anion radical, as shown in an equation (5). Then, the cytochrome c (divalent) resulting from the reduction is re-oxidized electrochemically by O2−., as shown in an equation (6), and an oxidizing current at that time is measured, thereby quantitatively detecting a concentration of the superoxide anion radical indirectly.cyt.c(Fe3+)+O2−.→cyt.c(Fe2+)+O2  (5)cyt.c(Fe2+)→cyt.c(Fe3+)+e−  (6)
However, the cytochrome c is an electron-transferring protein existing on a film of mitochondria within bio-cells and hence, to fabricate an electrode having the cytochrome c fixed thereon in an amount enough for the measurement, a large number of cells on the order of 105-106 are required, and there is a problem that the enzyme used is deactivated within several days. Therefore, it has been desired to develop an electrode which is capable of detecting active oxygen species such as a superoxide anion radical without need for a large amount of an enzyme and without the problem of the deactivation of the enzyme.
Therefore, the present applicant has proposed, in Japanese Patent Application No. 2000-387899, an reactive oxygen species electrode comprising a polymerized film of a metalloporphyrin complex formed on a surface of a conductive member, an active oxygen species concentration measuring sensor including such an active oxygen species electrode, a counter electrode and a reference electrode, and a process for detecting reactive oxygen species in a sample by measuring an electric current produced between a metal in the polymerized film of the metalloporphyrin and reactive oxygen species by the sensor.
This is based on that the electrode assembly comprising the polymerized film of the metalloporphyrin complex having a metal ion introduced into the center of a porphyrin compound, which film has been formed on the surface of the conductive member, is capable of detecting the presence and concentration of the reactive oxygen species without need for a large amount of an enzyme and without the deactivation problem.